From: Disher, Rose [CNTUS] <DisherR@Centocor.com>
  To  : ">"'Michael Morris'" , rasmb@alpha.bbri.org
  Date: Fri, 28 Apr 2000 10:51:19 -0400

RE: Incompetency number XXX

I've been following this all with great interest, and I am about to reveal
my social incompetence by being unable to resist an overused pun and
potentially stating the obvious. If you know, or have a good idea about, the
chemical nature or structure of the incompetent monomer, it is theoretically
possible to do a spiking study.  I don't remember with certainty, but I
don't recall any mention of this being done.  Chemical or enzymatic
modification to the competent species to produce the incompetent species
means that the spike supply is limited only by the amount of competent
material you have available.  The potential problem with this approach is
that you may or may not be able to make something which closely enough
approximates the incompetent species to be useful for modeling purposes.  A
way around this problem, however, is the fact that incompetent species are
often stability-indicating or the result of a reaction gone bad.  If you
know where or how the incompetent species is generated in the
expression/refold/purification/storage conditions, it is possible to
deliberately enrich for it, even if you don't know what it is.  If you can
spike in some incompetent-rich material, you can then follow the changes in
your data with the changes in spike concentration to sort out the cast of
characters in the centrifuge cell.

		-----Original Message-----
		From:	Michael Morris [michaelm@pharm.usyd.edu.au]">mailto:michaelm@pharm.usyd.edu.au]
		Sent:	Thursday, April 27, 2000 10:22 PM
		To:	rasmb@alpha.bbri.org
		Subject:	Incompetency number XXX

		Although you guys in the States are a day behind Australia
you are ahead in
		terms of business hours which means I come in and see a swag
of RASMB emails
		piled up on my server. So, I jump in on the end of the
discussion of
		incompetency for what it is worth.

		If you are sure the self-associating sample you have at
sedimentation
		equilibrium is uncontaminated by other proteins and
proteolytic breakdown
		products, then a sensible model-independent test for the
presence of
		incompetent species (incompetent monomer or irreversible
aggregates) is to
		look at the overlap (or lack of overlap) of weight-average
molecular weight
		plots or, much better, Omega plots. The model independence
of these simple
		tests is probably crucial since different K values might be
generated for
		each channel of data simply because the model being fitted
is wrong and not
		because incompetent species are present.

		Furthermore, the type of lack of overlap of the Omega or
Mw,app plots can
		provide useful clues as to whether the incompetent species
is monomeric or
		polymeric.

		If the type of incompetent species is known or can be
guessed there is no
		reason I can think of why it cannot be included in the model
to be fitted to
		the data. We have dabbled with this although never published
and I cannot
		think of any published examples.

		Michael Morris



	
__________________________________________________________________

		Michael Morris
		Senior Lecturer
		Pharmaceutical Chemistry	  Tel:	+ 61-2-9351-2359
		Department of Pharmacy A15	  Fax:	+ 61-2-9351-4391
		University of Sydney 2006	Email:
michaelm@pharm.usyd.edu.au 
		Australia			
	
__________________________________________________________________